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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 楊健志 | |
dc.contributor.author | Hsiao-Fang Chi | en |
dc.contributor.author | 紀曉芳 | zh_TW |
dc.date.accessioned | 2021-06-08T04:34:37Z | - |
dc.date.copyright | 2009-08-21 | |
dc.date.issued | 2009 | |
dc.date.submitted | 2009-08-18 | |
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Plant Cell 2: 673-684 Neuhaus JM, Sticher L, Meins F, Jr., Boller T (1991) A short C-terminal sequence is necessary and sufficient for the targeting of chitinases to the plant vacuole. Proc Natl Acad Sci U S A 88: 10362-10366 Ogata CM, Gordon PF, de Vos AM, Kim SH (1992) Crystal structure of a sweet tasting protein thaumatin I, at 1.65 A resolution. J Mol Biol 228: 893-908 Onishi M, Tachi H, Kojima T, Shiraiwa M, Takahara H (2006) Molecular cloning and characterization of a novel salt-inducible gene encoding an acidic isoform of PR-5 protein in soybean (Glycine max [L.] Merr.). Plant Physiol Biochem 44: 574-580 Ormo M, Cubitt AB, Kallio K, Gross LA, Tsien RY, Remington SJ (1996) Crystal structure of the Aequorea victoria green fluorescent protein. Science 273: 1392-1395 Prasher DC, Eckenrode VK, Ward WW, Prendergast FG, Cormier MJ (1992) Primary structure of the Aequorea victoria green-fluorescent protein. 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Plant Physiol 79: 126-137 Singh NK, Nelson DE, Kuhn D, Hasegawa PM, Bressan RA (1989) Molecular Cloning of Osmotin and Regulation of Its Expression by ABA and Adaptation to Low Water Potential. Plant Physiol 90: 1096-1101 Stintzi A, Heitz T, Prasad V, Wiedemann-Merdinoglu S, Kauffmann S, Geoffroy P, Legrand M, Fritig B (1993) Plant 'pathogenesis-related' proteins and their role in defense against pathogens. Biochimie 75: 687-706 van Loon LC, Rep M, Pieterse CM (2006) Significance of inducible defense-related proteins in infected plants. Annu Rev Phytopathol 44: 135-162 Velazhahan R, Zen KC, Muthukrishnan S (2001) Expression of a Rice Thaumatin-Like Protein (PR-5) Gene in a Baculovirus Expression System. Acta Phytopathologica et Entomologica Hungarica 36: 311-316 Yang F, Moss LG, Phillips GN, Jr. (1996) The molecular structure of green fluorescent protein. Nat Biotechnol 14: 1246-1251 Zhu B, Chen TH, Li PH (1995) Activation of two osmotin-like protein genes by abiotic stimuli and fungal pathogen in transgenic potato plants. Plant Physiol 108: 929-937 Zhu B, Chen TH, Li PH (1995) Expression of three osmotin-like protein genes in response to osmotic stress and fungal infection in potato. Plant Mol Biol 28: 17-26 Zimmer M (2002) Green fluorescent protein (GFP): applications, structure, and related photophysical behavior. Chem Rev 102: 759-781 劉麗飛. (2003) 生物技術方法--植物組織培養與基因轉殖實驗。國立臺灣大學生物技術研究中心 高艾玲. (2002) 阿拉伯芥中MAPR同源蛋白質基因之選殖、表現與功能分析。國立臺灣大學農業化學研究所碩士論文 范凱亭. (2005) 阿拉伯芥AtMAPRs之組織及細胞內定位探討。國立臺灣大學微生物與生化學研究所碩士論文 石依婷. (2008) 阿拉伯芥中Osmotin同源蛋白質的選殖與生理研究。國立臺灣大學微生物與生化學研究所碩士論文 | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/22944 | - |
dc.description.abstract | 菸草的Osmotin蛋白質會透過酵母菌細胞膜表面的接受體YOL002c,誘發酵母菌進行細胞凋亡。阿拉伯芥之HHP1 (heptahelical protein 1) 與YOL002c有高度序列保守性 (30% identity),皆為預測具有七個穿膜區塊的膜蛋白質。在過量表現osmotin的橄欖樹轉殖株中發現,較野生型有較多進行細胞凋亡的細胞。本篇研究目的在研究阿拉伯芥中的osmotin同源蛋白質AtOSM34與AtOLP在植物體的功能。利用pHBT95sGFP表現載體建構含 osmotin之綠色螢光融合蛋白質,於洋蔥表皮細胞與阿拉伯芥原生質體進行短暫表現,發現AtOSM34與AtOLP可能位於細胞質中。本研究建立AtOLP突變株,進一步探討其表現型是否與逆境相關。進行鹽逆境與水逆境處理後,發現AtOLP突變株在較高濃度鹽逆境與滲透壓逆境下,發芽率較野生型差,推論AtOLP可能與此逆境相關。本研究企圖建立以流式細胞儀觀察細胞週期,外加Osmotin處理阿拉伯芥原生質體,觀察是否誘發細胞凋亡。 | zh_TW |
dc.description.abstract | Osmotin from tobacco can induce apoptosis of yeast by binding to a yeast membrane protein, YOL002c. HHP1 (heptahelical protein 1) is a homolog (30% sequence identity) of YOL002c in Arabidopsis. Both HHP1 and YOL002c are predicted to have a seven-transmembrane domain. We were interested in whether or not osmotin can interact with HHP1 in Arabidopsis.
AtOSM34 and AtOLP are Arabidopsis proteins homologous to osmotin from tobacco. Transient expression of AtOSM34-sGFP and AtOLP-sGFP fusion proteins in onion epidermal cells and Arabidopsis protoplasts showed that osmotin are most likely to be located in cytosol. Heterologous protein expression of AtOSM34 and AtOLP in E. coli was established. T-DNA knock-down mutant of AtOLP was established using an ABRC line (WiscDsLox). We found that the mutant showed a slight decreasing in germination rate in high concentration of NaCl or mannitol. To determine whether or not osmotin could interact with HHP1, we established an apoptosis analysis method using flow cytometry with isolated nuclei. | en |
dc.description.provenance | Made available in DSpace on 2021-06-08T04:34:37Z (GMT). No. of bitstreams: 1 ntu-98-R96b47215-1.pdf: 3077862 bytes, checksum: f5c831ac941e07cccf0d8c0ed2627e4f (MD5) Previous issue date: 2009 | en |
dc.description.tableofcontents | 目錄....................................................................................................................................I
縮寫表…………………………………………………………………………….........IV 摘要……………………………………………………………………………….........VI Abstract……………………………………………………………………...………...VII 第一章 緒論……………………………………………………………..……...........1 1.1 PR5家族蛋白質…………………………………………………………....1 1.2 Osmotin相關功能之研究……………………………………………….....3 1.3 Osmotin like protein (OLP) 相關功能之研究…………………………….5 1.4 模式植物:阿拉伯芥 (Arabidopsis thaliana)……………………………..5 1.5 細胞定位中常用之報導基因……………………………………………...6 1.5.1 綠色螢光蛋白質GFP (Green fluorescent protein)…………………..6 1.6 研究動機、目的與方法…………………………………………………....7 第二章 材料與方法……………………………………………………………...9 2.1 實驗材料…………………………………………………………………...9 2.1.1 植物材料……………………………………………………………..9 2.1.2 載體 (vectors)………………………………………………………..9 2.1.3 大腸桿菌菌株……………………………………………………....10 2.2 實驗藥品………………………………………………………………….11 2.2.1 一般化學試劑……………………………………………………....11 2.2.2 酵素…………………………………………………………………11 2.2.3 培養基………………………………………………………………11 2.3 儀器設備………………………………………………………………….12 2.4 實驗方法………………………………………………………………….13 2.4.1 阿拉伯芥種植………………………………………………………13 2.4.1.1 培養基之配製………………………………………………...13 2.4.1.2 種子之表面消毒與低溫處理………………………………...15 2.4.1.3 種子之無菌培養……………………………………………...15 2.4.1.4 土壤培養……………………………………………………...16 2.4.1.5 收集種子……………………………………………………...16 2.4.2 DNA之抽取與分析….…………………………………………….17 2.4.2.1 阿拉伯芥染色體DNA之抽取………………………………17 2.4.2.2 質體DNA之抽取與分析……………………………………18 2.4.2.3 DNA限制酶切割分析方法………………………………….20 2.4.2.4 DNA瓊脂糖膠體電泳……………………………………….20 2.4.2.5 DNA片段的分離與純化……………………………………..21 2.4.2.6 DNA之去磷酸根反應………………………………………..21 2.4.2.7 DNA之磷酸化反應…………………………………………..21 2.4.2.8 DNA的定量…………………………………………………..22 2.4.3 RNA之抽取與分析……….………………………………………..22 2.4.3.1 Total RNA之抽取 (REzol reagent method).…………………..22 2.4.3.2 RNA的定量…….…………………………………………….23 2.4.3.3 甲醛瓊脂糖膠體電泳………………………………………..…..23 2.4.3.4 DNase treatment………………………………………………24 2.4.3.5 RT-PCR (reverse transcription polymerase chain reaction)…..25 2.4.4 AtOSM34、AtOLP基因序列選殖….……………………………...26 2.4.4.1 專一性引子設計……………………………………………...26 2.4.4.2 聚合酶鏈鎖反應 (Polymerase chain reaction)………………26 2.4.4.3 PCR反應產物之純化 (PCR clean up).……………………...27 2.4.4.4 T-A cloning……………………………………………………27 2.4.4.5 大腸桿菌之質體轉形………………………………………...28 2.4.4.6 重組質體的篩選……………………………………………...28 2.4.5 表現載體之建構…………………………………………………....29 2.4.5.1 暫時性基因表現 (transient expression) 載體之建構……....29 2.4.6 阿拉伯芥原生質體轉形……………………………………………30 2.4.6.1 原生質體抽取方法…………………………………………...30 2.4.6.2 原生質體基因轉形方法……………………………………...31 2.4.7 基因槍 (Particle bombardment) 轉殖……………………………..32 2.4.7.1 植物材料之準備……………………………………………...32 2.4.7.2 DNA包裹鎢粒子之製備…………………………………….32 2.4.7.3 操作基因槍…………………………………………………...33 2.4.8 顯微鏡及影像分析…………………………………………………34 2.4.9 AtOLP之T-DNA突變株篩選與分析.………..…………………...35 2.4.9.1 T-DNA突變株同型合子之確定……………………….…….35 2.4.9.2 AtOLP基因表現量之分析…………………………………...37 2.4.9.3 抑制AtOLP基因表現之定序確認…………………………..38 2.4.10 AtOLP突變株表現型之觀察……………………………………..38 2.4.10.1 處理滲透壓逆境AtOLP突變株發芽率之觀察…….………38 2.4.11 重組蛋白質之表現與檢定………………………………………..39 2.4.11.1 重組蛋白質最佳表現條件………………………………….39 2.4.11.2 重組蛋白質之大量表現…………………………………….40 2.4.11.3 重組蛋白之純化…………………………………………….40 2.4.11.4 Bradford蛋白質定量法……………………………………..41 2.4.11.5 電泳檢定法………………………………………………….42 2.4.12 蛋白質免疫轉印法………………………………………………..44 2.4.12.1 蛋白質電泳轉印法………………………………………….44 2.4.12.2 酵素免疫染色法…………………………………………….45 2.4.13 流式細胞儀之條件測試…………………………………………..46 2.4.13.1 外加AtOSM34或AtOLP蛋白質處理原生質體………….47 第三章 結果與討論……………………………………..………………………….48 3.1 AtOSM34與AtOLP基因序列選殖……………………………………...48 3.2 建構表現質體…………………………………………………………….48 3.3 AtOSM34與AtOLP之定位研究………………………………………...49 3.3.1 AtOSM34與AtOLP之序列比對與分析…………………………..49 3.3.2 洋蔥表皮定位分析…………………………………………………49 3.3.3 阿拉伯芥原生質體之定位分析……………………………………50 3.4 AtOLP之T-DNA突變株之篩選及觀察…………………………………51 3.4.1 Knockdown T-DNA突變株之篩選……….………………………..52 3.5 突變株於不同鹽濃度或金屬離子處理下之發芽率…………………….53 3.6 AtOSM34與AtOLP重組蛋白質之表現與分析..…………………..…...54 3.6.1 AtOSM34與AtOLP重組蛋白質最佳表現條件之探討…………..54 3.7 流式細胞儀之條件測試討論…………………………………………….55 第四章 結論與未來展望…………………………………………………………..57 4.1 結論……………………………………………………………………….57 4.2 未來展望………………………………………………………………….57 4.2.1 建構AtOSM34或AtOLP過度表現的轉殖株.……………….…..58 參考文獻……………………………………………………………………………..59 圖與表………………………………………………………………………………..64 附錄…………………………………………………………………………………..78 | |
dc.language.iso | zh-TW | |
dc.title | 阿拉伯芥中Osmotin同源蛋白質之細胞定位與突變株表型之分析 | zh_TW |
dc.title | Subcellular Localization of Osmotin Homologs in Arabidopsis thaliana and Mutant Phenotype Analysis | en |
dc.type | Thesis | |
dc.date.schoolyear | 97-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 蘇仲卿,王愛玉,李平篤,陳佩燁 | |
dc.subject.keyword | 阿拉伯芥,滲透壓,細胞定位,突變株, | zh_TW |
dc.subject.keyword | Arabidopsis,osmotin,subcellular,localization, | en |
dc.relation.page | 85 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2009-08-19 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 微生物與生化學研究所 | zh_TW |
顯示於系所單位: | 微生物學科所 |
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